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We analyzed cytokine expression in recipient spleens and cardiac allografts placed in mice that were unable to synthesize interleukin (IL)-4 due to disruption of the IL-4 gene (IL-4 -/-) and in wild-type (IL-4 +/+) mice. Polyclonal BL-4P and monoclonal 11B11, 1D11, and 24G2 anti-IL-4 antibodies were used to detect cell-surface and cytoplasmic antigens in sections of frozen tissue. All of the antibodies were found to react with non-IL-4 determinants associated with graft-infiltrating cells, and BL-4P, 1D11, and 24G2 bound to cells and connective tissue in the spleens of IL-4 -/- mice. The IL-4-producing cell line, X63Ag8-653 (X63), was used as a positive control for IL-4 staining and to test the ability of recombinant IL-4 to block the binding of antibodies to IL-4.

Successful pancreas transplantation requires the suppression of both allograft rejection and recurrence of autoimmune disease. In order to study treatments to suppress these two responses, separate models were developed for pancreas allograft rejection and autoimmune disease. In the first model, the diabetic state was induced with streptozotocin in CBA mice prior to the transplantation of pancreas grafts from BALB/c donors. In the absence of autoimmune disease, control mice rejected their grafts in 26 days (median). Antibody treatments (anti-lymphocyte serum and anti-CD4) significantly prolonged allograft survival beyond this time, but not to the extent we have previously reported in the heart graft model. NOD/Lt mice spontaneously developed autoimmune diabetes, and recurrence of disease was seen in isografts at 9.5 days (median). Antibody treatments significantly delayed disease recurrence, with anti-CD4 being the most effective. Heart allografts (CBA donors) in NOD/Lt recipients were rejected within 17 days (median), and the anti-CD4 treatment had a moderate effect in delaying graft survival (median 28 days). Anti-lymphocyte serum did not prolong graft survival. Thus antibody treatment was effective in delaying both rejection and the recurrence of autoimmune disease in segmental pancreas grafts. However, the same doses were not effective in delaying heart rejection in the NOD/Lt model, so it would appear that treatments which inhibit autoimmune disease may not prevent allograft rejection.

To examine the potential use of immunoconjugates of drugs and antibodies as immunosuppressive agents, mice were treated with a short course (4 days) of T-cell-specific anti-Ly-2.1 monoclonal antibody, or MAB conjugated to an anthracycline, idarubicin (IDA). The anti-Ly-2.1 MAB had no significant effect on the survival of BALB/c (Ly-2.2) heart allografts in CBA (Ly-2.1) mice, but was a potent immunosuppressive agent when coupled to IDA, with most grafts surviving for > 100 days following treatment with doses ranging from 10 to 120 micrograms IDA, covalently coupled to 1-8 mg MAB. IDA-MAB treated mice with long-surviving heart grafts showed donor-specific tolerance. They did not reject donor-type skin grafts (these survived for > 50 days), but rejected third-party skin in 10-14 days. Heart allografts in these mice survived for > 100 days. Allografts placed 30 days after treatment were rejected, showing a recovery of peripheral T cell function at this time. Newly derived thymic T cells were, however, not required for this recovery since adult thymectomized, IDA-MAB treated animals also recovered T cell function and rejected heart allografts. FACScan analysis of T cells from mice treated with 80 micrograms IDA-4 mg MAB, which had received a heart allograft, showed 95% T cell depletion in the spleen compared with ungrafted, IDA-MAB treated animals, and untreated controls with or without allografts. Splenic T cell depletion was however not significant in CBA mice immunosuppressed with the lower dose of 10 micrograms IDA-MAB. Thus rapid depletion of splenic T cells was not required for immunosuppression induced by IDA-MAB conjugates. However, the minor subpopulation of Ly-2+, which was activated by alloantigen while carrying IDA-MAB, may be depleted during the T cell response to the allograft, resulting in a state of alloantigen-specific tolerance in mice with long-surviving heart allografts.

We analyzed cytokine expression in recipient spleens and cardiac allografts placed in mice that were unable to synthesize interleukin (IL)-4 due to disruption of the IL-4 gene (IL-4 -/-) and in wild-type (IL-4 +/+) mice. Polyclonal BL-4P and monoclonal 11B11, 1D11, and 24G2 anti-IL-4 antibodies were used to detect cell-surface and cytoplasmic antigens in sections of frozen tissue. All of the antibodies were found to react with non-IL-4 determinants associated with graft-infiltrating cells, and BL-4P, 1D11, and 24G2 bound to cells and connective tissue in the spleens of IL-4 -/- mice. The IL-4-producing cell line, X63Ag8-653 (X63), was used as a positive control for IL-4 staining and to test the ability of recombinant IL-4 to block the binding of antibodies to IL-4.

In the NOD/Lt recipient mice, disease recurrence in untreated isografts was extremely rapid (median less than 10 days) compared to the rejection of an untreated BALB/c pancreas graft in a CBA mouse (median 26 days). This would be expected since disease recurrence is a secondary response in diabetic mice with lymphocytes primed to respond to the beta-cell autoantigen. The median survival time for the untreated CBA to NOD/Lt pancreas graft falls, as expected, between these two survival times (median 20 days). Although anti-CD4 and/or anti-CD8 were effective in delaying or stopping autoimmune disease recurrence and rejection in the separate models, they were unsuccessful in significantly altering survival times in the combined model, despite using 2-mg doses and dual therapy. Similar doses of anti-CD4 have failed to prevent islet allograft rejection in NOD/Lt mice. Long-term dual treatment may be required to inactivate CD4+ and CD8+ T cells in the NOD/Lt mouse to prevent both autoimmune disease recurrence and rejection. NOD/Lt recipients will require greater immunosuppression to prevent rejection-autoimmune disease recurrence will be easier to prevent. This study shows the value of using NOD/Lt mice, with naturally occurring type 1 diabetes, for assessment of immunosuppressive therapy to prevent failure of pancreas transplants.

In a mouse model for vascularized heart transplantation, CBA recipients of BALB/c hearts were treated with 0.25 mg of anti-CD4 (GK1.5) given intraperitoneally on the day of grafting and on days 1, 2, and 3 thereafter. This reduced splenic CD4+ cells to1% and all grafts survived100 days, compared with 8-10 days in untreated recipients. Despite recovery of the CD4+ cells after day 21, mice did not reject donor-type skin grafts at30 days, but rapidly rejected third-party skin, showing alloantigen-specific tolerance. The surviving heart grafts had significant mononuclear cell infiltration at time points from 7 to 100 days after transplantation. In the normal rejection process, where extensive myocyte necrosis was seen at 7 days, graft-infiltrating T cells produced IL-2 and IFN-gamma. These cells responded in vitro to IL-2 and displayed donor-specific CTL activity. In contrast, cells from CD4-mAb-treated hearts did not show significant growth in IL-2 or kill donor cells in CTL assays. In these nonrejecting hearts, immunohistology showed a diffuse infiltrate of T cells and macrophages by day 3. The allograft infiltrate increased rapidly thereafter in both rejecting and nonrejecting grafts, peaking at day 6-7 in rejecting grafts, when CD4+, CD8+, and IL-2R+ cells were present, with expression of IL-2, IFN-gamma, and IL-4, but only trace levels of IL-10. From 14 to 100 days, nonrejecting allografts showed a characteristic cytokine profile of dense IL-4 and IL-10 expression on intragraft leukocytes and endothelial cells, with low levels of IL-2 and IFN-gamma. This cytokine profile, characteristic of Th2 responses, was seen in all nonrejecting grafts and was not present in rejecting grafts. Allograft tolerance can studied by examination of the functions and cytokine profile of the cells within the graft, and tolerance develops in the presence of a Th2 response within the graft.

The blacksmith Chromis punctipinnis, an abundant planktivorous damselfish off southern California, USA, shelters along rocky reefs at night. While sheltered, blacksmiths excrete ammonium that could, in turn, be utilized by nearby benthic macrophytes. Laboratory experiments during the summer and fall of 1983 and 1984 indicate that ammonium excretion at night ranged from 18.1 μmol h-1 by a 8.5 g (dry) fish, to 89.1 μmol h-1 by a 27.3 g fish; excretion rates generally declined throughout the night. Field measurements at night indicate that ammonium concentrations were significantly higher in rocky crevices occupied by blacksmiths than in unoccupied shelters, and the ammonium level in one shelter dropped after a blacksmith was experimentally removed. Young kelp plants (Macrocystis pyrifera) are capable of taking up ammonium at night. Ammonium levels in chambers containing both a blacksmith and a young kelp plant were significantly lower than in chambers containing only a fish, and ammonium levels dropped in ammoniumspiked chambers that contained kelp plants. Nighttime ammonium uptake rates by young kelp plants, which averaged 1.6 μmol g-1 (dry) h-1, were only slightly lower than those during the day. Daytime excretion by blacksmiths occasionally results in elevated ammonium levels in the water column. On two of six days, ammonium concentrations in midwater foraging aggregations were slightly but significantly higher than in upcurrent controls; since blacksmiths typically aggregate at the incurrent margin of kelp beds, the ammonium is swept downcurrent and may be utilized by large M. pyrifera that extend through the water column. Thus, the activities of blacksmiths may results in the importation of extrinsic, inorganic nitrogen to primary producers on temperate reefs.